Primary transcripts in higher eukaryotes contain intervening sequences which must be precisely excised to generate functional messenger RNAs. Nuclear pre-mRNA splicing is thus an essential step in regulating gene expression in every eukaryotic cell. Regulated and alternative splicing events play a role in determining normal cell development and generate a broad spectrum of genetic diversity in higher eukaryotes. Aberrant splicing is associated with certain diseases; for example, disruption of splicing patterns has been implicated in the oncogenic activation of c-Hras, one of the most commonly mutated genes in human cancer. Although much progress has been made in defining the general features of splicing as well as identifying specific components, understanding the regulation of this process will require the analysis of the splicing machinery on the molecular level. Elucidation of RNA-RNA and RNA-protein interactions and the way in which conformational changes are achieved in the spliceosome is central to this understanding. Insights into these questions can be gained by studying the molecular interactions of ATPases, such as Prp16, known to function at specific steps of splicing. This proposal presents experiments to delineate the mechanisms by which Prp16 uses ATP hydrolysis to promote the final step of the splicing reaction leading to the formation of mature RNA. We propose to dissect the second step by reconstituting the partial reactions from purified components. The choice of yeast as the experimental system permits the powerful combination of genetic and biochemical approaches in studying the molecular interactions in complex processes. Given the large degree of evolutionary conservation between yeast and mammals in the structure and function of the basic splicing apparatus, our studies will be broadly relevant to pre-mRNA splicing in higher eukaryotes.